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Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
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JoVE Journal 生化学
Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

DOI:
10.3791/62112-v

•

09:53 min

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February 07, 2021

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, , , ,
1化学科,Tufts University, 2物理学科,Wesleyan University, 3Wyss Institute at Harvard University

章

  • 00:00Introduction
  • 00:31Preparation of Quantum-Dot-Labeled DNA Substrate Materials
  • 03:38Microfluidic Device Construction
  • 05:58Surface Tethering of Quantum-Dot-Labeled DNA Substrate
  • 07:07REase-Mediated DNA Cleavage
  • 07:51Results: Single-Molecule TIRF Experiment for Parallelized Observations of DNA Cleavage Events
  • 09:32Conclusion

概要

自動翻訳

自動翻訳

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

タグ

Single-molecule Dwell-time AnalysisRestriction EndonucleaseDNA CleavageMultiplexed ApproachGamma Probability DistributionOligonucleotideDTTQuantum DotSulfo-SMCCCHES BufferPBS

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